Fluorescence Anisotropy Decay Kinetics Research Articles

Supramolecular assembly of coumarin 7 with sulfobutylether-β-cyclodextrin for biomolecular applications.

Coumarins, in general, exhibit a wide range of photophysical characteristics and are highly sensitive to their microenvironment, and, therefore, their fluorescence characteristics have attracted immense attention as sensors in chemical and biological systems. In the present study, the supramolecular interaction of a bichromophoric coumarin dye, namely, Coumarin 7 (C7) with sulfobutylether-β-cyclodextrin (SBE7βCD) macrocyclic host at different pH conditions has been investigated by using optical spectroscopic techniques such as absorption, steady-state and time-resolved emissions, and circular dichroism measurements and compared with that of βCD. Considerable enhancement in the fluorescence intensity and lifetime of C7 on complexation with SBE7βCD proposes that non-radiative processes like TICT behavior are strictly hindered due to the confinement in the host cavity experienced by the C7 dye. The increase in the rotational correlation time evaluated from the fluorescence anisotropy decay kinetics further confirms the formation of tightly bound inclusion complexes. The binding constant values reveal that the monocationic form of dye at pH 3 shows ∼3 times stronger interaction with SBE7βCD than the neutral form of dye at pH 7 due to strong electrostatic cation-anion interaction. SBE7βCD:C7 exhibits an improved photostability and an upward pK a shift of 0.4 unit compared to the contrasting downward pK a shift of 0.5 with the βCD. The enhanced fluorescence yield and increased photostability have been exploited for bioimaging applications, and better images were captured by staining the Drosophila fly gut with the SBE7βCD:C7 complex. The enhancement in the binding interaction and the emission intensity were found to be responsive to external stimuli such as small competitive binders or metal ions and nearly quantitative dissociation of the complex was demonstrated to release the dye and would find stimuli-responsive applications.

Site-Specific Fluorescence Dynamics To Probe Polar Arrest by Fob1 in Replication Fork Barrier Sequences.

Fob1 protein plays an important role in aging and maintains genomic stability by avoiding clashes between the replication and transcription machinery. It facilitates polar arrest by binding to replication fork barrier (RFB) sites, present within the nontranscribed spacer region of the ribosomal DNA. Here, we investigate the mechanism of unidirectional arrest by creating multiple prosthetic forks within the RFB, with fluorescent adenine analogue 2-aminopurine incorporated site-specifically in both the “permissible” and “nonpermissible” directions. The motional dynamics of the RFB-Fob1 complexes analyzed by fluorescence lifetime and fluorescence anisotropy decay kinetics shows that Fob1 adopts a clamp-lock model of arrest and causes stronger perturbation with the bases in the double-stranded region of the nonpermissible-directed forks over those of the permissible directed ones, thereby creating a polar barrier. Corroborative thermal melting studies reveal a skewed distribution of GC content within the RFB sequence that potentially assists in Fob1-mediated arrest.

Molecular crowding causes narrowing of population heterogeneity and restricts internal dynamics in a protein

Macromolecular crowding is a distinguishing property of intracellular media. Knowledge on the structure and dynamics of a protein in a crowded environment is essential for a complete understanding of its function. Reduction in intermolecular space could cause structural and functional alterations. Here, we have studied a model protein barstar to see how polyethylene glycol (PEG)-induced crowding affects its various structural states (native, unfolded and molten-globule-like) with different extents of change in conformational heterogeneity. Intramolecular distances and distance distributions were determined by time-resolved Förster resonance energy transfer from Trp53 to several acceptor sites by analysis of fluorescence decay kinetics using the Maximum Entropy Method. We observed PEG-induced narrowing of population distributions along with shifting of populations towards more compact states. Structural compactness also resulted in the slowing down of internal dynamics of the protein as revealed by fluorescence anisotropy decay kinetics of the fluorophore IAEDANS attached at several sites.

Carboxymethylation of Cysteines Impedes Aggregation of Hen Lysozyme in Alkaline pH

In our previous work, we have shown that hen lysozyme spontaneously forms globular and fibrillar aggregates which are stabilized later through formation of intermolecular disulphide bond in pH 12.2. Therefore, focus of the present work was on suppression of HEWL aggregation by carboxymethylation of free -SH groups with iodoacetamide that inhibits further formation of disulphide bonds. Changes in structure and dynamics of aggregates were monitored using various biophysical techniques like fluorescence spectroscopy and atomic force microscopy (AFM). Thioflavin T (ThT) fluorescence showed that in comparison to free thiol containing HEWL control, carboxymethylated HEWL was unable to form fibrils. However presence of moderate ThT intensity in modified HEWL indicated presence of oligomers. Increased fluorescence intensity and marginal red shift of ANS spectra in modified HEWL compared to unblocked control revealed that modified HEWL possessed exposed hydrophobic residues compared to free -SH containing HEWL. Fluorescence anisotropy of carboxymethylated dansyl-labeled HEWL were significantly lower compared to control. The fluorescence anisotropy decay kinetics revealed that carboxymethylated HEWL-dansyl conjugates possessed shorter global rotational correlation time (∼11.7 ns) compare to control (∼22 ns) after 30 hours of incubation in pH 12.2 at room temperature. Taken together, these investigations suggest that blocking thiol groups in HEWL suppresses formation of amyloid fibrils and bigger aggregates but not oligomers, in pH 12.2 at room temperature. This work can be helpful for food industries in making aggregate-free lysozyme preservatives in future.Authors acknowledge Department of Science and Technology, New Delhi and University Grants Commision New Delhi for financial support.

Site-Pecific Fluorescnce Dynamics in an RNA ‘THERMOMETER’ Reveals the Mechanism of Temperature-Sensitive Translation

The ROSE (Repression Of heat Shock gene Expression) element of mRNA present in the 5’-UTR of small heat-shock genes in many Gram-negative bacteria is known to function as a ‘RNA thermometer’ by controlling protein translation in a temperature range of 30 - 42° C where the translation is blocked till 30°C and allowed at 42°C and beyond, perhaps due to an unfolding transition of the ROSE hair-pin motif.In this work, we have used site-specific fluorescence labeling and pico-second time-domain fluorescence spectroscopy to unravel the mechanism. The ‘ROSE RNA’ was site-specially labeled with 2-aminopurine (2-AP), a fluorescent analog of adenine. Observables such as fluorescence lifetime, fluorescence anisotropy decay kinetics and dynamic fluorescence quenching revealed properties such as the level of base stacking, rotational motion of the bases, segmental dynamics of the backbone and the level of exposure of base to solvent. As expected, all read-outs of 2-AP residue that were studied showed remarkable position-dependence/sensitivity in the RNA sequence at 25°C. The striking result was the persistence of the same position-dependence of the parameters even at 45°C albeit at a measurably reduced levels. However the same position-dependence was nearly ‘wiped out’ in the presence of urea where all intra-molecular interactions in RNA are undone. These observations have prompted us to revise the existing model of ROSE RNA action: we now suggest that unlike proposed earlier, the thermometer action of ROSE emanates not from its unfolding structural transition between 25 and 45°C, but rather from its propensity to enhance structural dynamics without “melting” the structure. We hypothesize that either the enhanced dynamics of the structure it self or its full melting due to an extrinsic factor (perhaps a protein interaction) might be the basis of its thermometer action.

Exploration of the Correlation between Solvation Dynamics and Internal Dynamics of a Protein

Protein function is intimately related to the dynamics of the protein as well as to the dynamics of the solvent shell around the protein. Although it has been argued extensively that protein dynamics is slaved to solvent dynamics, experimental support for this hypothesis is scanty. In this study, measurements of fluorescence anisotropy decay kinetics have been used to determine the motional dynamics of the fluorophore acrylodan linked to several locations in a small protein barstar in its various structural forms, including the native and unfolded states as well as the acid and protofibril forms. Fluorescence upconversion and streak camera measurements have been used to determine the solvation dynamics around the fluorophore. Both the motional dynamics and solvent dynamics were found to be dependent upon the location of the probe as well as on the structural form of the protein. While the (internal) motional dynamics of the fluorophore occur in the 0.1-3 ns time domain, the observed mean solvent relaxation times are in the range of 20-300 ps. A strong positive correlation between these two dynamical modes was found in spite of the significant difference in their time scales. This observed correlation is a strong indicator of the coupling between solvent dynamics and the dynamics in the protein.

Characterization of the Heterogeneity and Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of Site-Specific Fluorescence Anisotropy Decay Kinetics

The aggregation of proteins often results in highly ordered fibrillar structures. While significant insights have been obtained on structural aspects of amyloid fibrils, little is known about the structures of protofibrils, which are presumed to be the precursors of fibrils. An understanding of the molecular mechanism of the formation of protofibrils and fibrils requires information on the landscape of interpeptide interactions. This work addresses this question by using, as a model protein, barstar, which forms protofibrils and fibrils at low (< 3) pH. Use was made of the heterogeneity of aggregate populations encountered during fibril formation. Population heterogeneity was scored through rotational dynamics monitored by time-resolved fluorescence anisotropy of an environment-sensitive fluorophore, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), attached to specific locations in the protein. Firstly, it was observed that barstar, when labeled at certain locations with 1,5-IAEDANS, did not form mixed protofibrils with the corresponding unlabeled protein. Labeled and unlabeled proteins formed protofibrils as separate populations. A two-population model of fluorescence anisotropy decay kinetics exhibiting a ‘dip-and-rise’ behavior was the main readout in arriving at this conclusion. Additional support for this conclusion came from the fluorescence lifetime of the probe 1,5-IAEDANS. Subsequently, the location of the fluorophore was moved along the length of the protein in nine mutant proteins, and the capability to form mixed fibrils was assessed. The results revealed that about two-thirds of the protein sequence at the C-terminal end of the protein was intimately involved in the formation of ordered protofibrils, probably forming the core, while the remaining one-third of the protein (i.e., the N-terminal region) remained largely noninteractive and flexible. This methodology can be used as a general strategy to identify regions of a given protein sequence involved in interprotein interactions in amyloid protofibrils.

Fluorescence Characterization of the Structural Heterogeneity of Polytene Chromosomes

Studies on the physical nature of the structural heterogeneity of chromatin in their native states are few. The eukaryotic chromatin as observed by dye staining studies is of heterogeneous intensity when observed by fluorescent stains, where less and more bright regions apparently correspond to euchromatin and heterochromatin respectively. These are also associated with differential gene expression where it is believed that euchromatin is transcriptionally more active due to increased flexibility. Unfixed squashed preparations of polytene chromosomes of Drosophila were stained with a dsDNA specific dye PicoGreen and fluorescence lifetimes as well as fluorescence anisotropy decay kinetics were measured. Here we report a positive correlation between fluorescence lifetimes and fluorescence intensities, and show that less bright regions corresponding to euchromatin have shorter lifetimes, whereas more bright regions corresponding to heterochromatin have longer lifetimes. We interpret this as less bright regions being more dynamic, a conclusion also supported by fluorescence anisotropy decay kinetics. We infer that the comparatively higher flexibility associated with euchromatin can be directly measured by fluorescence lifetimes and fluorescence anisotropy decay kinetics.

Evidence for Chain Length Dependent Local Ordering Surrounding Perylene Dissolved in Poly(ethylene glycol)

Time-resolved fluorescence anisotropy measurements on dilute solutions of perylene dissolved in poly(ethylene glycol) (PEG) oligomers are carried out as a function of oligomer chain length and temperature. The perylene fluorescence anisotropy decay kinetics are best described by a double-exponential decay law. The temperature dependence of the perylene rotational reorientation dynamics in PEG is exponential activated; however, at the lower PEG chain lengths, the activation energies that describe the perylene rotational reorientation are significantly larger than the activation energy for PEG viscous flow. As the PEG molecular weight increases, the activation energies associated with the perylene rotational motions become equivalent to the activation energy for PEG viscous flow. The principal perylene rotational diffusion coefficients (D∥ and D⊥) are also a strong function of PEG chain length and temperature. The ratio of the principal diffusion coefficients (D∥/D⊥) shows that D⊥ is hindered relative to D∥ as the PEG chain length increases. These results are complementary to recent work from Goldie and Blanchard on the rotational reorientation dynamics of perylene dissolved in n-alkanols. On the basis of our data, we propose a chain length dependent ordering of the PEG oligomers around the perylene molecules that impedes D⊥ to a greater extent compared to D∥.

Selective steady-state and time-resolved fluorescence spectroscopy of an HLA-A2-peptide complex

The human class I major histocompatibility complex (MHC) encoded molecule HLA-A2 loaded with the high-affinity peptide GILGRVFTL (p790), was studied by means of steady-state and picosecond fluorescence intensity and fluorescence anisotropy methods. The large number of tryptophan residues (W) (10 W/heavy chain, 2 W β 2 m ) as well as their fluorescence sensitivity to the microenvironment, determine the emission of the studied complex. The HLA-A2/peptide complex exhibits a considerable static inhomogeneous broadening (IB) of the W electronic spectra, which results in a dependence of the steady-state fluorescence spectrum on the excitation wavelength. The high concentration of W's chromophores and the spectral IB cause a directed non-radiative migration of electronic excitation energy by Foerster's mechanism from ‘blue’ W residues to ‘red’ ones. This phenomenon manifests itself in a nanosecond fluorescence spectral shift and an accelerated fluorescence depolarization at the red edge of the emission spectrum. Selective excitation at the red edge of the W absorption band (310 nm) provided a space selective reduction in the number of excited chromophores and enabled resolution of the emission of the ‘red’ subset of the protein's tryptophans. This avoided the non-radiative homo-energy transfer and enabled to study the fluorescence anisotropy decay kinetics of these residues without a distortion by the energy transfer (ET) process. Under these experimental conditions the fluorescence anisotropy decays practically from the limiting anisotropy value (0.3) for W in a bi-exponential process. The longer decay constant has a value larger than that expected for a global rotation of the HLA-A2/ peptide complex suggesting that the protein molecules exist in an oligomeric form. No clear assignment for the fast component can currently be made; it may be either a manifestation of a limited internal rotation of the W residues or a result of uncompleted compensation of the homo-ET process.

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

Abstract—The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T1,≅0.8 ns, T2≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a/Zea complex, in which Chl a/Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl‐proteins of the Chi alb light‐harvesting complex of plant photosynthesis.